Biological Clones Research Articles

Resolution of multiclonal infections of Trypanosoma cruzi from naturally infected triatomine bugs and from experimentally infected mice by direct plating on a sensitive solid medium

The isolation of biological clones of Trypanosoma cruzi by microscopically dispensing individual organisms or by serial dilution is laborious and time consuming. The inability to resolve mixed T. cruzi infections, from vectors and hosts, and to isolate clones of slow growing genotypes by efficient plating on solid media, has hindered characterisation studies and downstream applications. We have devised and validated a sensitive, solid medium plating technique for rapid in vitro isolation of clones representative of all the recognised T. cruzi lineages (TCI, TCIIa–e), including the slow growing strain CANIII (TC IIa) and Trypanosoma rangeli, with high plating efficiencies. Furthermore, the method is effective for the isolation of clones directly from silvatic triatomine bugs and from experimentally infected mice harbouring mixed infections, allowing resolution of multiclonal infections from varied sources.

Dynamics of Mutation and Recombination in a Replicating Population of Complementing, Defective Viral Genomes

In a previous study, we documented that serial passage of a biological clone of foot-and-mouth disease virus (FMDV) at high multiplicity of infection (moi) in cell culture resulted in viral populations dominated by defective genomes that included internal in-frame deletions, affecting the L and capsid-coding regions, and were infectious by complementation. In the present study, analyses of the defective genomes present in individual viral plaques, and of consensus nucleotide sequences determined for the entire genomes of sequential samples, have revealed a continuous dynamics of mutation and recombination. At some points of high genetic instability, multiple minority genomes with different internal deletions co-existed in the population. At later passages, a new defective RNA arose and displaced a related, previously dominant RNA. Nucleotide sequences of the different genomic forms found in sequential isolates have revealed an accumulation of mutations at an average rate of 0.12 substitutions per genome per passage. At the regions around the deletion sites, substantial, minor or no nucleotide sequence identity is found, suggesting relaxed sequence requirements for the occurrence of internal deletions. Competition experiments indicate a selective advantage of late phase defective genomes over their precursor forms. The defective genome-based FMDV retained an expansion of host cell tropism, undergone by the standard virus at a previous stage of the same evolutionary lineage. Thus, despite a complex dynamics of mutation and recombination, and phases of high genetic instability, a biologically relevant phenotypic trait was stably maintained after the evolutionary transition towards a primitive genome segmentation. The results extend the concept of a complex spectrum of mutant genomes to a complex spectrum of defective genomes in some evolutionary transitions of RNA viruses.

Isolation of human immunodeficiency virus-type 1 (HIV-1) clones with biological and molecular properties of the primary isolate

We developed a new biological cloning system for HIV-1 isolates using the U87.CD4 cell lines that express different chemokine receptors. We demonstrate that our method is sensitive and specific because the clones isolated had the same coreceptor usage and genotype as viruses of the primary isolate. We evaluated our cloning system by isolating 27 biological clones from two primary HIV-1 R3R5X4 isolates. Three HIV-1 phenotypes (R3R5X4, R3R5 and R5) were identified in isolate 29 and two (R3R5X4 or R5X4) in isolate 31. Each phenotype was distinguished by a unique genotype. Sequencing of 20 molecular clones from each isolate did not reveal additional genotypes. One of the three genotypes identified from isolate 29 was not found by molecular cloning of the original isolate, suggesting high specificity and sensitivity of the biological cloning system in isolating minor virus populations. Our results suggest that the new cloning approach can be used as an alternative to the existing method for isolating biological clones in PBMC.

Genetic basis for GPI-anchor merozoite surface antigen polymorphism of Babesia and resulting antigenic diversity

Glycosyl-phosphatidylinositol anchor merozoite surface antigens (GPI-anchor MSA) are proposed to act in the invasion process of infective merozoites of Babesia into host erythrocytes. Because of their essential function in the survival of Babesia parasites, they constitute good candidates for the development of vaccines against babesiosis and they have been extensively analyzed. These include Babesia bovis variable MSA (VMSA) and Babesia bigemina gp45/gp55 proteins of the agents of bovine babesiosis from tropical and subtropical countries, and the Babesia divergens Bd37 and Babesia canis Bc28 proteins of the main agents of bovine and canine babesiosis in Europe, respectively. However, these are very polymorphic antigens and Babesia parasites have evolved molecular mechanisms that enable these antigens to evade the host immune system as a survival strategy. This review focuses on the genetic basis of GPI-anchor MSA polymorphism and the antigenic diversity of B-cell epitopes that might be generated in each of these Babesia species. The picture is incomplete and no Babesia genome sequence is yet available. However, the available sequences suggest that two distinct, non cross-reactive GPI-anchor MSA (i.e., with unique B-cell epitopes) may be required by all Babesia species for invasion, and that these two distinct GPI-anchor MSA would be encoded by a multigene family. Furthermore, the data are consistent with the ability of biological clones from Babesia to use these multigene families for the expression of GPI-anchor MSA, either conserved ( B. canis and B. bovis) or polymorphic ( B. divergens and B. bigemina) in their amino acid sequence. Moreover, as a consequence for successful parasitism, the data suggest that both conserved and polymorphic GPI-anchor MSA would present unique B-cell epitopes.

Replicative fitness of CCR5-using and CXCR4-using human immunodeficiency virus type 1 biological clones

CCR5-tropic viruses cause the vast majority of new HIV-1 infections while about half of the individuals infected with HIV-1 manifest a co-receptor switch (CCR5 (R5) to CXCR4 (X4)) prior to accelerated disease progression. The underlying biological mechanisms of X4 outgrowth in AIDS patients are still poorly understood. Although X4 viruses have been associated with increased “virulence” in vivo, in vitro replication and cytopathicity studies of X4 and R5 viruses have led to conflicting conclusions. We studied the replicative fitness of HIV-1 biological clones with different co-receptor tropism, isolated from four AIDS patients. On average, R5 and X4 clones replicated equally well in mitogen-activated T cells. In contrast, X4 variants were transferred more efficiently from dendritic cells to autologous CD4 + T cells. These observations suggest that interaction between X4 viruses, DC and T cells might contribute to the preferential outgrowth of X4 viruses in AIDS patients.

CTL escape and increased viremia irrespective of HIV-specific CD4 + T-helper responses in two HIV-infected individuals

We investigated whether development of mutations leads to loss of CD8 T-cell recognition in HIV-1 infection and is possibly linked to alterations in HIV-1-specific CD4 + T-cell responses in 2 HIV-infected individuals. In patient, H434 full genome sequencing of HIV-1 biological clones at early and late time points during disease progression showed development of fixed mutations in 16 predicted HIV-specific CTL epitopes. Loss of T-cell recognition and reactivity against wild-type and mutant epitopes was observed primarily for the HLA-B27-restricted KK10 epitope and HLA-A2-restricted SL9 epitope. Similarly, in patient H671, decreasing numbers of HLA-A3-restricted CD8 + T cells specific for the wild-type RK9 epitope was observed after CTL escape. Only in patient H434 loss of CTL responses was paralleled by a decrease in HIV-specific IL-2 + CD4 + T-helper responses. This suggests that loss of T-cell reactivity may not be directly linked to HIV-specific CD4 + T-cell responses but that increased viremia after CTL escape may influence CD4 + T-helper responses.

Current ethical problems in cell biology

Summary We selected important ethical conflicts of interest in current cell biology and which remain still unresolved: the use of human biological material, patents for biological material, cloning and use of stem cells, genetic engineering, onset of human life and death, and the use of vertebrate biomodels. New data from cell and molecular biology cannot facilitate the solution of such problems – on the contrary, they make these solutions more difficult. A solution may be found within the socio-cultural problems. Intensive research is necessary both to accomplish economical and medical benefit and to clarify ethical rules. Thus, essential cell biology seems to be the important part of education for non-life sciences students and students of biology and medicine would touch the social and ethical implications of recent biotechnologies in each knowledge-based economy.

Following the very initial growth of biological RNA viral clones

Due to their extremely high genetic diversity, which is a direct consequence of high mutation rates, RNA viruses are often described as molecular quasispecies. According to this theory, RNA virus populations cannot be understood in terms of individual viral clones, as they are clouds of interconnected mutants, but this prediction has not yet been demonstrated experimentally. The goal of this study was to determine the fitness of individual clones sampled from a given RNA virus population, a necessary previous step to test the above prediction. To do so, limiting dilutions of a vesicular stomatitis virus population were employed to isolate single viral clones and their initial growth dynamics were followed, corresponding to the release of the first few hundred viral particles. This technique is useful for estimating basic fitness parameters, such as intracellular growth rate, viral yield per cell, rate at which cells are infected and time spent in cell-to-cell transmission. A combination of these parameters allows estimation of the fitness of individual clones, which seems to be determined mainly by their ability to complete infection cycles more quickly. Interestingly, fitness was systematically higher for initial clones than for their derived populations. In addition to environmental changes, such as cellular defence mechanisms, these differences are attributable to high RNA virus mutation rates.

R5 Human Immunodeficiency Virus Type 1 Infection of Fetal Thymic Organ Culture Induces Cytokine and CCR5 Expression

Late-stage CCR5 tropic human immunodeficiency virus type 1 (HIV-1) isolates (R5 HIV-1) can deplete nearly all CD4+ thymocytes from human thymus/liver grafts, despite the fact that fewer than 5% of these cells express CCR5. To resolve this paradox, we studied the replication and cytopathic effects (CPE) of late-stage R5 HIV-1 biological clones from two progressors and two long-term nonprogressors (LTNP) in fetal thymic organ culture (FTOC) with and without added cytokines. We found that R5 HIV-1 clones from progressors but not LTNP were cytopathic in untreated FTOC. Moreover, R5 HIV-1 clones from progressors replicated to higher levels than LTNP-derived R5 HIV-1 clones in this system. In contrast, when FTOC was maintained in the presence of interleukin 2 (IL-2), IL-4, and IL-7, both progressor and LTNP clones exhibited similar replication and CPE, which were equal to or greater than the levels achieved by progressor-derived R5 HIV-1 clones in untreated FTOC. This finding was likely due to IL-2-induced CCR5 expression on CD4+ thymocytes in FTOC. R5 HIV-1 clones showed greater pathogenesis for CCR5+ cells but also showed evidence of CPE on CCR5- cells. Furthermore, infection of FTOC by R5 HIV-1 induced IL-10 and transforming growth factor beta (TGF-beta) expression. Both IL-10 and TGF-beta in turn induced CCR5 expression in FTOC. Induction of CCR5 expression via cytokine induction by R5 HIV-1 infection of CCR5+ thymocytes likely permitted further viral replication in newly CCR5+ thymocytes. CCR5 expression, therefore, is a key determinant of pathogenesis of R5 HIV-1 in FTOC.

Elements of developmental biology

Developmental biology has differentiated into a rich, complex field that continues to progress at breathtaking speed. This presents a conundrum for textbook authors: present students with a comprehensive survey of the entire field or distill it down to the essence that provides students with a

Monitoring Sequence Space as a Test for the Target of Selection in Viruses

An essential feature of viral quasispecies, predicted from quasispecies theory, is that the target of selection is the mutant distribution as a whole. To test molecularly the mutant composition selected from a viral quasispecies we reconstructed a mutant distribution using 19 antigenic variants of foot-and-mouth disease virus (FMDV). Each variant was marked by a specific amino acid replacement at a major antigenic site of the virus that conferred resistance to a monoclonal antibody (mAb). The variants were introduced in the mutant spectrum of a biological FMDV clone, at a frequency commonly found in FMDV quasispecies. The reconstructed quasispecies (and a number of control populations) were allowed to replicate in the presence or absence of the mAb. The mutant distribution that became dominant as a result of antibody selection included at least ten of the 19 mutants initially used to reconstruct the quasispecies. No such biased mutant repertoire was found in control populations. The results show that a mutant distribution was selected, and are incompatible with selection of an individual genome, which then generated multiple mutants upon further replication. An ample representation of variants immediately following a selection event should contribute to subsequent adaptability of the virus.

Virological phenotype switches under salvage therapy with lopinavir-ritonavir in heavily pretreated HIV-1 vertically infected children.

To investigate the effects of salvage therapy with lopinavir-ritonavir on HIV-1 phenotype in heavily antiretroviral experienced HIV-infected children. Twenty antiretroviral experienced HIV-infected children were studied during a mean of time of 16.1 months from initiation of the treatment with lopinavir-ritonavir. Besides CD4 T cells, viral load and clinical status, we analyzed 91 serial viral isolates to study the phenotype, and biological clones derived from co-cultivation techniques. We observed an increase in CD4 T cells, a statistically significant decrease in viral load and clinical benefits from 3 months after treatment. Ninety per cent of children had SI/X4 bulk isolates in peripheral blood mononuclear cells at study entry. The viral phenotype changed to non syncitium-inducing (NSI)/R5 in 94% of the children after a mean of 5.7 months (95% confidence interval, 2.1-9.3 months) of salvage therapy. The remaining 10% of children had NSI/R5 isolates at entry and at all follow-up study. Similar results were found at the clonal level. Thus, at study entry in PBMC of three children with bulk syncitium-inducing (SI) phenotype, we recovered 65 biologic clones, 56 being SI and nine NSI. After salvage therapy bulk isolates changed to NSI and of 40 biologic clones recovered only five were SI and the rest were NSI. Our data suggest that lopinavir-ritonavir salvage therapy led not only to a viral load decrease but also to a phenotypic change. X4 virus appeared to be preferentially suppressed. Shifts in co-receptor usage may thereby contribute to the clinical efficacy of anti-HIV drugs in vertically infected infants.

Identification of Sequential Viral Escape Mutants Associated with Altered T-Cell Responses in a Human Immunodeficiency Virus Type 1-Infected Individual

Control of viremia in natural human immunodeficiency virus type 1 (HIV-1) infection in humans is associated with a virus-specific T-cell response. However, still much is unknown with regard to the extent of CD8(+) cytotoxic T-lymphocyte (CTL) responses required to successfully control HIV-1 infection and to what extent CTL epitope escape can account for rises in viral load and ultimate progression to disease. In this study, we chose to monitor through full-length genome sequence of replication-competent biological clones the modifications that occurred within predicted CTL epitopes and to identify whether the alterations resulted in epitope escape from CTL recognition. From an extensive analysis of 59 biological HIV-1 clones generated over a period of 4 years from a single individual in whom the viral load was observed to rise, we identified the locations in the genome of five CD8(+) CTL epitopes. Fixed mutations were identified within the p17, gp120, gp41, Nef, and reverse transcriptase genes. Using a gamma interferon ELIspot assay, we identified for four of the five epitopes with fixed mutations a complete loss of T-cell reactivity against the wild-type epitope and a partial loss of reactivity against the mutant epitope. These results demonstrate the sequential accumulation of CTL escape in a patient during disease progression, indicating that multiple combinations of T-cell epitopes are required to control viremia.

Decreasing sensitivity to RANTES (regulated on activation, normally T cell-expressed and -secreted) neutralization of CC chemokine receptor 5-using, non-syncytium-inducing virus variants in the course of human immunodeficiency virus type 1 infection.

In approximately half of human immunodeficiency virus (HIV) type 1-infected individuals, the development of CXC chemokine receptor 4-using, syncytium-inducing (SI) virus variants precedes a rapid progression to acquired immunodeficiency syndrome (AIDS). In other individuals, only CC chemokine receptor 5-using (R5), non-SI (NSI) virus variants are present throughout infection. These individuals may be either long-term survivors (LTSs) or rapid progressors. The basis for this variable disease progression in individuals with only R5 virus variants is not yet fully understood. In this study, the beta-chemokine sensitivity of biological HIV-1 clones isolated from 13 individuals who harbored only R5, NSI virus variants (7 LTSs and 6 progressors) was investigated. We found a statistically significant decrease in sensitivity of virus variants to RANTES (regulated on activation, normally T cell-expressed and -secreted) neutralization during the course of progressive infection, but not during follow-up of LTSs. Our data suggest that a role exists for RANTES neutralization sensitivity of HIV-1 in AIDS pathogenesis.

A live attenuated equine infectious anemia virus proviral vaccine with a modified S2 gene provides protection from detectable infection by intravenous virulent virus challenge of experimentally inoculated horses.

Previous evaluations of inactivated whole-virus and envelope subunit vaccines to equine infectious anemia virus (EIAV) have revealed a broad spectrum of efficacy ranging from highly type-specific protection to severe enhancement of viral replication and disease in experimentally immunized equids. Among experimental animal lentivirus vaccines, immunizations with live attenuated viral strains have proven most effective, but the vaccine efficacy has been shown to be highly dependent on the nature and severity of the vaccine virus attenuation. We describe here for the first time the characterization of an experimental attenuated proviral vaccine, EIAV(UK)deltaS2, based on inactivation of the S2 accessory gene to down regulate in vivo replication without affecting in vitro growth properties. The results of these studies demonstrated that immunization with EIAV(UK)deltaS2 elicited mature virus-specific immune responses by 6 months and that this vaccine immunity provided protection from disease and detectable infection by intravenous challenge with a reference virulent biological clone, EIAV(PV). This level of protection was observed in each of the six experimental horses challenged with the reference virulent EIAV(PV) by using a low-dose multiple-exposure protocol (three administrations of 10 median horse infectious doses [HID(50)], intravenous) designed to mimic field exposures and in all three experimentally immunized ponies challenged intravenously with a single inoculation of 3,000 HID(50). In contrast, naïve equids subjected to the low- or high-dose challenge develop a detectable infection of challenge virus and acute disease within several weeks. Thus, these data demonstrate that the EIAV S2 gene provides an optimal site for modification to achieve the necessary balance between attenuation to suppress virulence and replication potential to sufficiently drive host immune responses to produce vaccine immunity to viral exposure.

Characterization of EIAV LTR variability and compartmentalization in various reservoir tissues of long-term inapparent carrier ponies

Dynamic genomic variation resulting in changes in envelope antigenicity has been established as a fundamental mechanism of persistence by equine infectious anemia virus (EIAV), as observed with other lentiviruses, including HIV-1. In addition to the reported changes in envelope sequences, however, certain studies indicate the viral LTR as a second variable EIAV gene, with the enhancer region being designated as hypervariable. These observations have lead to the suggestion that LTR variation may alter viral replication properties to optimize to the microenvironment of particular tissue reservoirs. To test this hypothesis directly, we examined the population of LTR quasispecies contained in various tissues of two inapparent carrier ponies experimentally infected with a reference EIAV biological clone for 18 months. The results of these studies demonstrated that the EIAV LTR is in fact highly conserved with respect to the infecting LTR species after 1.5 years of persistent infection and regardless of the tissue reservoir. Thus, these comprehensive analyses demonstrate for the first time that the EIAV LTR is highly conserved during long-term persistent infection and that the observed variations in viral LTR are associated more with in vitro adaptation to replication in cultured cells rather than in vivo replication in natural target cells.

Mechanism of genetic exchange in American trypanosomes.

The kinetoplastid Protozoa are responsible for devastating diseases. In the Americas, Trypanosoma cruzi is the agent of Chagas' disease--a widespread disease transmissible from animals to humans (zoonosis)--which is transmitted by exposure to infected faeces of blood-sucking triatomine bugs. The presence of genetic exchange in T. cruzi and in Leishmania is much debated. Here, by producing hybrid clones, we show that T. cruzi has an extant capacity for genetic exchange. The mechanism is unusual and distinct from that proposed for the African trypanosome, Trypanosoma brucei. Two biological clones of T. cruzi were transfected to carry different drug-resistance markers, and were passaged together through the entire life cycle. Six double-drug-resistant progeny clones, recovered from the mammalian stage of the life cycle, show fusion of parental genotypes, loss of alleles, homologous recombination, and uniparental inheritance of kinetoplast maxicircle DNA. There are strong genetic parallels between these experimental hybrids and the genotypes among natural isolates of T. cruzi. In this instance, aneuploidy through nuclear hybridization results in recombination across far greater genetic distances than mendelian genetic exchange. This mechanism also parallels genome duplication.

Analysis, quantification, and evolutionary consequences of HIV-1 in vitro recombination.

HIV-1 recombination was studied in vitro by viral cocultivation of four combinations of strains of subtypes B, D, and F. Viral cocultivations were performed in MT-4 cells and maintained for 22 days. The parental and recombinant forms were quantified by a specific PCR system in an env fragment of 2500 nucleotides. On day 5, there was a close correlation between the proportion of recombination and the genetic distance between strains. In three of the four viral combinations studied, a steady increase in the proportion of recombinant genomes was observed over time. This rise coincided with the progressive loss of one of the parental strains, resulting in less diverse viral populations. Nucleotide sequencing of biological recombinant clones from the B/D cocultivation revealed a higher number of recombination events in pol than in env gene, and an increasing number of crossovers per clone with time.

Adaptive Changes after Human Immunodeficiency Virus Type 1 Transmission

Primary HIV-1 infection (PHI) is associated with a period of viremia, the resolution of which generally coincides with the development of both humoral and cellular immune responses. In this study replication-competent quasispecies were derived from virus isolated from an individual before and after seroconversion. Virus was also isolated from the presumed donor. Phenotypic and genotypic analysis of biological clones identified transmission of an R5/M-tropic phenotype. However, the ability of clones derived from the recipient to replicate in primary macrophages and PBMCs was restricted after transmission. This apparent selection process was supported by analysis of molecular clones derived from the isolated virus. Analysis of the ratio of synonymous and nonsynonymous substitutions predicted the existence of selective pressure soon after transmission, coincident with the development of HIV-1-specific antibodies. An Env trans-complementation assay demonstrated that the infectivity of a clone derived from the recipient after seroconversion was enhanced in the presence of a selected neutralizing antibody, indicating that the developing humoral immune response may have at least in part contributed to the selective pressure identified.

In vitro analysis of human immunodeficiency virus type 1 resistance to nevirapine and fitness determination of resistant variants.

Nevirapine-resistant variants were generated by serial passages in MT-2 cells in the presence of increasing drug concentrations. In passage 5, mutations V106A, Y181C and G190A were detected in the global population, associated with a 100-fold susceptibility decrease. Sequence analysis of biological clones obtained from passage 5 and subsequent passages showed that single mutants, detected in first passages, were progressively replaced in passage 15 by double mutants, correlating with a 500-fold increase in phenotypic resistance. Fitness determination of single mutants confirmed that, in the presence of nevirapine, every variant was more fit than wild-type with a fitness order Y181C>V106A>G190A>wild-type. Unexpectedly, in the absence of the drug, the Y181C resistant mutant was more fit than wild-type, with a fitness gradient Y181C>wild-type >G106A>or=V190A. Using a molecular clone in which the Y181C mutation was introduced by in vitro mutagenesis, the greater fitness of the Y181C mutant was confirmed in new competition cultures. These data exemplify the role of resistance mutations on virus phenotype but also on virus evolution leading, occasionally, to resistant variants fitter than the wild-type in the absence of the drug.